Team:UCLA/Notebook/Protein Cages/16 July 2015

Intro: Today, Philip and I ran the PCR reaction to amplify our PCQuad and add our histags in the end to fit our DNA into the vector with a T7 and RBS along with iGEM restriction sites. Once the PCR was done, we ran in it on a gel to verify if the pcr worked and if we were getting appropriate band.

Made working stock of 20 ul of 1ng/ul of Template DNA. Resuspended primers and made working stock of 10uM.

PCR Protocol

5.0  microliter of Q5 buffer 
2.5  microliter of 2uM dNTPs
1.25 microliter of 10uM forward primer 
1.25 microliter of 10uM reverse primer 
.25 microliter of template DNA
.25 microliter of Polymerase 
16.5 microliter of DD Water.

Total Volume 27 microliter.

Gradient Temperatures: 66,62,58. 62,66 worked the best.

Gel Making

1g of Agarose 
Add 100 mL of 1x TAE
Microwave for 1-3 min(30s interval check)
Cool until able to handle then add sybr Safe(10ul)
Pour gel into gel caster 
Leave at room temp until solid.(Turn of light when casting gel).

Gel Protocol

Add 5ul(of 6x) of loading buffer to all each sample 
place gel in box 
Fill Box with 1X TAE buffer.
Load ladder into first lane.(5ul)
Load samples.
Run gel at 100V for 20 minutes( make sure gel runs from (-)to (+) with loading side facing (-). 

Left to Right 58,62,66

-Nithin